DISINFECTANT VALIDATION

DISINFECTANT VALIDATION

To evaluate the efficiency of disinfectants that are being used for the surface and area sanitization of controlled and critical clean rooms. The disinfectants when used as per the recommendations of the manufacturer for dilution and the contact time should be able to inhibit the growth of the test microorganisms.

TABLE OF CONTENTS

• Protocol Approval
• Objective
• Acceptance Criteria
• Responsibilities
• Validation Methodologies
• Evaluation of Test Result
• Re-validation Frequency
• Documentation
• Conclusion

PROTOCOL APPROVAL 

• Name / Designation
• Prepared By
• Microbiologist
• Checked By
• Microbiology
• Manager. Quality Control
• Checked By
• Microbiology
• Manager. Quality Control
• Approved By
• Plant Manager
• Manager. Quality Assurance

OBJECTIVE:

To evaluate the efficiency of disinfectants that are being used for the surface and area sanitization of controlled and critical clean rooms.

ACCEPTANCE CRITERIA:

The disinfectants when used as per the recommendations of the manufacturer for dilution and the contact time should be able to inhibit the growth of the test microorganisms.

RESPONSIBILITIES:

The Validation Group consists of the following members:

• QA/QC Department Representative
• Manager, QA
• Manager QC
• Manager, Microbiology
• Microbiologists
• Specific Responsibilities
• The microbiologist will write the protocol in consultation with the QC Manager.
• Manager, QA will check the protocol for its completeness, accuracy, technical excellence, and applicability.
• Head of the Department QA will be responsible for the final approval of the protocol.
• Microbiologists will be responsible for the preparation of slants, subculturing of organisms, and streaking on the slants.
• A microbiologist will also be responsible for preparing the used dilution as per the manufacturer’s recommendation.

VALIDATION TEST PROCEDURE:

Before proceeding for validation following materials are required.

• Staphylococcus aureus
• B.Subtilis
• E.coli.
• Candida albicans
• Aspergillus niger.

Micrococcus species Environmental Isolate

• Disinfectants for Validation.
• Sterile distilled water.
• Sterile Molten Soyabean Casein Digest Agar
• Sterile Molten Potato Dextrose Agar
• Poured SCDA plates.
• Poured PDA plates
• Sterile forceps
• Sterile membrane filtration units.
• Sterile membranes
• Vortex Mixer.

Preparation of Spore Forming Culture

1. Prepare SCDA slants as per the SOP.
2. Incubate slant for 48 hrs. at 30 – 35 ° C as pre-incubation to cross-check the contamination.
3. From the working culture streak a loopful of Bacillus subtilis culture on to the newly prepared slant.
4. Incubate the slants at 30 – 35 ° C for 48 hrs.
5. After 48 hrs. of incubation cross check the culture for any cross-contamination by simple gram staining technique.
6. During Gram Staining also check for the presence of vegetative or spore cells.
7. After Gram Staining preserve the cultures for further 7 days.
8. After 7 days cross check the culture for any spore formation by simple Negative Staining technique.

Negative staining.

1. Prepare a thin smear on a clean slide.
2. Place the slide on a staining rack above boiling water. Over the smear with small pieces of tissue paper.
3. Pour malachite green and continue heating till boiling for 5 minutes.
4. Do not allow the stain to dry.
5. Gently wash the slide with water.

Preparation of Challenge Inoculum.

1. Prepare fresh SCDA /PDA slants as per SOP.
2. Incubate those slants at 30 – 35 ° C for 48 hrs. to cross-check any type of contamination.
3. From the working culture streak a loopful of culture into the freshly prepared slants as per the SOP.
4. Incubate those inoculated slants at 30 – 35 ° C for 48 hrs. for bacterial cultures and 20 – 25 ° C for 5 days for fungal cultures.
5. Add 5.0 ml of sterile saline into the slants aseptically taking care not to contaminate the slants.
6. With the help of a sterile spatula streak the lawn of organisms.
7. Take 1.0 ml of the culture suspension and make serial dilutions ranging from 1:10 to 1: 100.
8. Plate the serial dilutions from 1:10 to 1:100 taking 1.0 ml of the culture in sterile petri dishes.
9. Pour sterile molten SCDA and PDA onto the inoculated plates.
10. Incubate the plates at the required specified temperature.
11. After plating the required dilutions do not discard the dilutions and preserve the samples at a temperature of 2 – 8 ° C till the incubation period.
12. After the incubation count the number of colonies.
13. Select the dilution which is having 10000 to 100000 cells/ml for validation study.
14. Preserve the culture suspension. Record the data in Annexure. 

Preparation of Disinfectant solution

Take the concentrated solution as received from the supplier and dilute the disinfectant to prepare a stock solution, which is twice that of the recommended concentration. This will be the used dilution.

Determining the initial microbial count.

1. With the help of a calibrated micropipette pipette out 1.0 ml of any of the cultures and inoculate into 9.0 ml of the 0.9 % sterile saline solution.
2. Vortex it for 5.0 minutes.
3. Filter the sample through a 0.45 m membrane.
4. Give three washings of 100 ml each with .1 % sterile peptone water.
5. After filtration, with the help of sterile forceps take the membrane and place the membrane on a SCDA or m (HPC) agar plate.
6. Incubate the plate.
7. After incubation count the number of colonies present on the membrane.
8. Note down the number of colonies.
9. This will be the initial counts of the culture.
10. In the same manner, proceed for all the other cultures which are going to be tested.
11. Record the initial counts in the Annexure and report them as Counts / 0.1 ml

Determining the Efficacy of the Disinfectant by Use Dilution Method.

1. Prepare test tubes having 9.0 ml of sterile distilled water.
2. Add 1.0 ml of the use dilution for one disinfectant.
3. Vortex the tube for 5.0 minutes.
4. Add 0.1 ml of any one culture into the test sample.
5. Make the sample dilution in such a way that each contact time has two sets of samples.
6. Give a contact time of 0 hrs. 5.0 min, 10 min.
7. After the specified contact time, filter the samples through a 0.45 m membrane.
8. Give three washings of 100 ml each with .1 % sterile peptone water.
9. After filtration with the help of sterile forceps take the membrane and place it on an SCDA or m (HPC) agar.
10. Incubate the plates.
11. After incubation count the number of colonies present on the membrane.
12. Note down the number of colonies.
13. This will be the final count of the exposed culture.
14. Select the plates that have the least nil counts.
15. Proceed in the same manner taking all the cultures to be tested.
16. Contact time for the usage of the disinfectant will be set based on the results which will have the least counts. 

Determining the Efficacy of the Disinfectant by Surface Method

1.  To get a practical approach for the efficacy apply the disinfectants to any of the surfaces which is present in that particular area which will be decontaminated by spraying the disinfectant.
2.  Take S.S. strips and epoxy-coated material having a surface area of 25 cm^2.
3.  Wrap the strips and the epoxy-coated surface with Aluminum foil and sterilize it in a Dry heat Sterilizer at 200 ⁰ C for 2 hrs.
4. From the previously determined suspension having 10000 – 100000 cells per ml inoculate one culture onto three different S. S surface and Epoxy coated surfaces.
5. With the help of a sterile spatula spread the culture on the surface.
6. Keep it on the LAF bench for 30 minutes for drying.
7. After the exposed duration for drying spray the disinfectant onto any one surface of the recommended used dilution.
8. Allow the surface to be with the sanitizer for 0hrs, 5.0 min, and 10 min.
9. With the help of a sterile moistened swab, swab the surface gently covering all the area of the surface.
10. Use different swabs for all the strips.
11. Place the two swab sticks in a test tube with having sterile solution of fluid casein digest Soya broth.
12. Vortex the test tube gently for 5. 0 minutes.
13. Aseptically filter the samples through a 0.45 m membrane.
14. Give three washings of 100 ml each with .1 % sterile peptone water.
15. After the filtration with the help of sterile forceps take the membrane 5.52 and place it on a SCDA or m (HPC) agar.
16. Incubate the plates.
17. After incubation count the number of colonies present on the membrane.
18. Note down the number of colonies in both the test sample and that with the unexposed strip.
19. Proceed in the same manner taking all the cultures to be tested and the various sanitizers. 

EVALUATION OF RESULTS:

The decrease in the bacterial load to the exposed disinfectant indicates that the disinfectant is capable of reducing the contaminant when used in the area.

Schedule Revalidation:

Revalidation shall be carried out in case of

• A new disinfectant is received.
• If the manufacturer revises the concentration of ingredients.
• If a new microorganism is isolated from that particular area. 

DOCUMENTATION:

The validation Report contains the following documents.

• Approved Validation protocol
• Media Preparation as per SOP.
• Gram Staining GTP.
• Test report for different disinfectants
• List of Approved Disinfectants.
• Summary Conclusion.

CONCLUSION:

A summary report will contain a discussion and conclusion which clearly state the achievement of the objective of validation studies.

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